| Pathogens can escape from the host immune assault by antigen mask and blockage antibody, and they can live in the host and result in chronic infection. Most recently, regulatory T cells have been reported to be involved in immune evasion of pathogens. Many parasites including Leishmania major can result in the clustering of regulatory T cells, which favors the pathogen to survive while the antibody blocking regulatory T cells contributes to parasite killing.Regulatory T cells were firstly described by Sakaguchi et al in 1995, which were divided into natural and induced regulatory T cells. Natural regulatory T cells are originated from thymus and maintain in periphery, which constitutively expressed markers of CD25, Foxp3, CTLA-4 and GITR. CD25 molecule, IL-2 receptor a band, is also expressed on the surface of activated effector cells, while Foxp3 is important for the suppressive function and is specific for CD4+CD25+ Tregs. CD4+CD25+ Tregs can suppress the activation of CD4+ and CD8+ effector T cells by direct contact with CD4+CD25+ Tregs and effector T cells or suppressive cytokines of IL-10 and TGF-0. CTLA-4 (cell cytotoxic T lymphocyte associated-antigen), a co-inhibitory receptor, is correlated with the host immune tolerance. Combination treatment of blockage antibody or treatment alone favors for the host clearing parasites.Studies on vaccines against Schistosoma japonicum (S. japonicum), including dead vaccine, attenuated live vaccine, genetically engineering and nucleic acid vaccine, have often yielded disappointing results. Gluthatione-S-transferase (GST) is one of the candidated vaccines against S. japonicum recommended by WHO, but not usually acquires 40% protective efficacy. Since CD4+CD25+ Tregs can suppress effector T cells, the poor protective efficacy of vaccine against S. japonicum may be related to CD4+CD25+ Tregs and blockade antibody can enhance the protective efficacy of vaccine.In this study, one hand, we explore the relationship between CD4+CD25+ Tregs and infection with S. japonicum and the effect and mechanism of blockade antibody on the protective efficacy against S. japonicum. On the other hand, we explore the relationship between GST vaccine against S. japonicum and CD4+CD25+ Tregs and the effect and mechanism of anti-CD25 mAb on the protective efficacy of GST vaccine against S. japonicum.This study was divided into two parts:Part 1:The effect and mechanism of CD4+CD25+ Tregs on the immune evasion of S. japonicum.Objective:To explore the effect and mechanism of CD4+CD25+ Tregs on the immune evasion of S. japonicum and the effect of S. japonicum infection and anti-CD25 mAb on the percentages of CD4+CD25+ Tregs.Methods:Animal experiments include three parts Part 1:BALB/c female mice were randomly divided into 2 groups:normal and infected group. Each mouse in infected group was infected with 40 S. japonicum cercarie. Mice in two groups were sacrificed at 2,3,4 and 5 weeks after infection respectively. Flow cytometry was performed to detect the percentage of CD4+CD25+ Tregs in spleen cells.Part 2:BALB/c female mice were randomly divided into 2 groups:normal, and anti-CD25 mAb group. Each mouse in anti-CD25 mAb group were injected intraperitoneally 300μg of anti-CD25 mAb and that of control group was injected equal volume of PBS. All mice were succumbed at 2 weeks after treatment. Spleens were obtained sterile and prepared single cell suspension. Flow cytometry was performed for detection of the frequency of CD4+CD25+ Tregs.Part 3:BALB/c female mice were randomly divided into 2 groups, infected control and anti-CD25 mAb group. Each mouse were infected 40 S. japonicum cercarie. Mice were injected intraperitoneally 300μg anti-CD25 mAb at 2 weeks after infection, and equal volume PBS as control. All mice were sacrificed at 6 weeks after infection to measure worm burden and eggs number in liver, reduction rate of worm and reduction rate of eggs per gram liver was evaluated. Spleens were obtained sterile and prepared single cell suspension. The levels of cytokines in spleen cell cultural suprernatant were detected by sandwich ELIS A after stimulation by ConA.Results:1) At 2,3,4 and 5 weeks after infection, the percentage of CD4+CD25+ Tregs were prompted from 1.99±0.10% to 2.36±0.37%,2.8±0.05%,2.35±0.09% and 2.68±0.05% respectively. It demonstrats that S. japonicum infection can induce the expansion of CD4+CD25+ Tregs.2) The percentage of CD4+CD25+ Tregs in normal group was 1.90±0.10%, which was 0.20±0.05% at 2 weeks post-CD25 mAb administration. It demonstrats that anti-CD25 mAb can partially block CD4+CD25+ Tregs.3) The reduction rate of worm burden at 3 weeks post-anti-CD25 mAb was 18.99% and the reduction rate of eggs of per gram liver was 15.86%. It demonstrates that the reduction of CD4+CD25+ Tregs favors the clearance of S. japonicum in the host body. 4) The levels of IFN-γand IL-5 were increased by anti-CD25 mAb administration.Conclusion:Anti-CD25 mAb favors the host to clear S. japonicum by reducing CD4+CD25+ Tregs.Part 2:The effect and mechanism of CD4+CD25+ Tregs on the protective efficacy of GST vaccine against S. japonicumObjective:To explore the effect and mechanism of CD4+CD25+ Tregs on the protective efficacy of GST vaccine against S. japonicom by the effect of vaccine GST and anti-CD25 mAb on the expression of CD4+CD25+ Tregs.Methods:BALB/c female mice were randomly divided into 5 groups:normal group, infected control group, anti-CD25 mAb group, GST group and co-treated group with anti-CD25 mAb and GST. Each mouse in GST group and co-treated group was injected 50μg of GST each mouse at 2-week interval,3 times immunization totally. At 2 weeks after the last immunization, each mouse was infected with 40 S. japonicum cercarie. Each mouse in anti-CD25 mAb and co-treated group were injected intraperitoneally 300μg of anti-CD25 mAb at 2 weeks after infection and equal volume of PBS was as control. Four groups six each were sacrificed at 2,3,4 and 5 weeks after infection respectively. Flow cytometry was performed to detect the percentage of CD4+CD25+ Tregs. The levels of cytokines in spleen cell cultural supernatant were detected by sandwich ELISA. Worm burden and eggs in per gram liver were valuated at 5 weeks after infection. Liver tissues were stained with HE to detect egg granuloma.Results:1) The percentages of CD4+CD25+ Tregs in infected control and GST group were 2.36±0.37% and 3.36±0.06% at 2 weeks after infection respectively; The percentages of CD4+CD25+ Tregs in infected control and GST group were 2.8±0.05% and 2.97±0.08% at 3 weeks after infection; 2.35±.09% and 2.47±0.09% at 4 weeks after infection; 2.68±0.05% and 3.03±0.13% at 5 weeks after infection respectively. It demonstrates that GST vaccine can induce the expansion of CD4+CD25+ Tregs.2) The percentages of CD4+CD25+ Tregs were decreased at 1 week post-anti-CD25 mAb (3 weeks after infection) from 2.8±0.05% to 0.13±0.04%. The percentages of CD4+CD25+ Tregs in infected control and anti-CD25 mAb group were 2.35±0.09% and 0.9±0.23% at 2 weeks post-anti-CD25 mAb administration (4 weeks after infection); 2.68±0.05% and 1.93±0.03% at 3 weeks post-anti-CD25 mAb administration (5 weeks after infection) respectively. It demonstrates that CD4+CD25+ Tregs can be blocked at 1 week after anti-CD25 mAb administration, which was partially restored but still lower than those in infected control group at 2 and 3 weeks post-anti-CD25 mAb adminstration.3) The reduction rate of worm burden and eggs per gram liver in GST group was 24.98% and 32%, which were prompted to 43.43% and 49% in co-treated group respectively. It demonstrates that anti-CD25 mAb can enhance the protective efficacy of GST vaccine as an adjuvant.4) Thl type immune response is predominant in the early stage of S. japonicum infection, which shifts to Th2 immune response at 4 weeks after infection. Thl type cytokine IFN-y was still sustain at higher level by the administration of anti-CD25 mAb and favored the host to clear S. japonicum. It demonstrates that anti-CD25 mAb enhances the protective efficacy of GST vaccine by increasing Thl type immune response.5) Pathogenical analysis denoted that there were no significant differences in the size and infiltrating cells of egg granuloma in each infected group. It demonstrates that anti-CD25 mAb can not enhance the pathogenical injury.Conclusion:GST vaccine against S. japonicum can induce the frequency of CD4+CD25+ Tregs which influence the protective efficacy of GST; anti-CD25 mAb can partially block CD4+CD25+ Tregs and enhance Thl type immune response which can enhance the protective efficacy of GST. So anti-CD25 mAb can act as an adjuvant to enhance the protective efficacy of GST vaccine against S.japoncium.In conclusion, this study demonstrated:1) CD4+CD25+ Tregs are involved in parasites immune evasion, and the expansion of CD4+CD25+ Tregs favors parasites escaping from the host immune assault.2) It is the first time that GST vaccine against S. japonicum can induce CD4+CD25+ Tregs on the infected background and CD4+CD25+ Tregs are involved in the poor protective efficacy of GST vaccine.3) It is the first time that anti-CD25 mAb can enhance the protective efficacy of vaccine GST against S. japonicum by blocking CD4+CD25+ Tregs and enhancement Thl type immune response.4) It is the first time that anti-CD25 mAb can as an adjuvant to enhance the protective efficacy of GST vaccine against S. japoncium. |
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